Thrombin binds to the highly anionic fibrinogen γ' chain through anion-binding exosite II. This binding profoundly alters thrombin's ability to cleave substrates, including fibrinogen, factor VIII, and PAR1. However, it is unknown whether this interaction is due mainly to general electrostatic complementarity between the γ' chain and exosite II or if there are critical charged γ' chain residues involved. We therefore systematically determined the contribution of negatively charged amino acids in the γ' chain, both individually and collectively, to thrombin binding affinity. Surface plasmon resonance binding experiments were performed using immobilized γ' chain peptides with charged-to-uncharged amino acid substitutions, i.e., Asp to Asn, Glu to Gln, and pTyr to Tyr. Individually, the substitution of uncharged for charged amino acids resulted in only minor changes in binding affinity, with a maximal change in K(d) from 0.440 to 0.705 μM for the Asp419Asn substitution. However, substitution of all three charged amino acids in a conserved β-turn that is predicted to contact thrombin, pTyr418Tyr, Asp419Asn, and pTyr422Tyr, resulted in the loss of measurable binding, as did substitution of all the flanking charged amino acids. In addition, the binding of the γ' chain to thrombin was weakened in a dose-dependent manner with increasing NaCl concentration, resulting in a net loss of three or four ion pairs between thrombin and the γ' chain. Therefore, although each of the individual charges in the γ' chain contributes only incrementally to the overall binding affinity, the ensemble of the combined charges plays a profound role in the thrombin-γ' chain interactions.